RNA interference (RNAi), a potent and selective gene silencing mechanism, has revolutionized the field of biological science. The ability of RNAi to specifically down-regulate the expression of any gene has had a profound impact on the study of gene function, and holds great promise for in vivo functional genomics. The current paradigm for inducing RNAi in mammalian cells relies on the use of a 21-bp siRNA structural scaffold. This siRNA scaffold, which consists of a 19-bp duplex with 2-nt overhangs at each 3'end, has been widely used for in vitro gene function studies, as well as for the development of novel therapeutics for various human diseases. Despite the extensive use of siRNA for in vitro studies, this technology is plagued by variable gene silencing effects, the activation of innate immune responses, and the knockdown of non-targeted mRNA (off-target silencing). These well-recognized effects complicate the interpretation of gene function experiments that rely on specific target gene knockdown. Moreover, these problems can become impossible to manage in large scale functional genomics experiments that test gene function in vivo. Boston Biomedical Inc. has discovered aiRNA (asymmetric interfering RNA), a novel proprietary technology for inducing highly efficient RNAi in mammalian cells. aiRNA has shown superior in vitro gene silencing properties compared to siRNA. Moreover, aiRNA completely abolished sense-strand off-target silencing, and eliminated or significantly reduced interferon response induction. These findings indicate that aiRNA can meet the urgent need for a technology to selectively knockdown target genes, and suggest that aiRNA holds significant potential for broad applications in biological research, including functional genomics. The long-term goal of this SBIR proposal is to optimize aiRNA as a reagent for gene function research, including genome-wide functional genomics studies. In Phase I we will perform studies to optimize the structure and design of aiRNA gene silencers that can be used by biomedical researchers. In Aim 1, we will examine various aiRNA structures to determine the best design for inducing potent and highly specific silencing under various experimental conditions. In Aim 2, we will examine relevance of current siRNA design algorithms for devising efficacious aiRNA. In Aim 3, we will perform experiments to identify key elements for the development of an aiRNA-specific design algorithm. Our overall goal in Phase II will be to develop a genome-wide validated aiRNA reagent for functional genomics research. These studies will entail the construction of a library based on the optimized aiRNA structures (developed in Phase I) against all known human genes. The ability of the aiRNA library to mediate efficacious and specific gene silencing in vitro will then be evaluated. The further development of aiRNA technology should significantly improve our ability to analyze gene function, and may have significant implications for other applications involving RNAi-mediated gene silencing. PUBLIC HEALTH RELEVANCE: The current paradigm for inducing RNA interference (RNAi) in mammalian cells relies on the use of short interfering RNA (siRNA) that have been used extensively for in vitro gene silencing;however, various non- specific effects relating to the use of siRNA have been recognized as a significant drawback to their use in gene function research. Boston Biomedical Inc. has discovered aiRNA (asymmetric interfering RNA), a novel proprietary technology for inducing highly efficient RNAi in mammalian cells that has superior in vitro gene silencing properties, eliminated sense-strand mediated off-target silencing, and markedly reduced or eliminated non-specific interferon-like response induction compared to conventional siRNA. The research outlined in this proposal is designed to optimize the structure of, and develop a design algorithm for, aiRNA gene silencers that can be used by the biomedical research community, with the long-term goal of developing this technology as a reagent for genome-wide functional genomics studies.